Ubiquitin-like proteins are receiving increasing attention due to their recently discovered roles in diverse biological processes. One such protein, named Smt3 (also known as Sumo or Sentrin), has a wide range of suspected functions, including functions in transcriptional regulation, nuclear transport, and cytokinesis. Like ubiquitin, Smt3 becomes covalently attached to target proteins via isopeptide linkages to lysine residues. These targets include transcription factors, components of the nuclear pore complex, and components of the septin complex. While many Smt3-conjugation targets have been identified, studies of Smt3 function in metazoans has largely been limited to cell culture analysis. This proposal describes a combined biochemical, cell biological, and genetic analysis of Smt3-conjugation in Drosophila, with the goal being to determine the functional roles of this process in the developing organism. The specific aims are to: (1) Identify targets of Smt3-conjugation in Drosophila by expressing tagged forms of Smt3 in vivo and then using the tags to purify Smt3-conjugates from protein extracts; (2) Determine the results of perturbing Smt3-conjugation in cultured Drosophila cells on such processes as protein localization and transcriptional regulation; (3) Determine the developmental roles of Smt3-conjugation in the intact organism by using genetic and reverse genetic approaches to perturb Smt3-conjugation in vivo, with the goal of making direct links between observed phenotypes and specific targets of Smt3-conjugation. These studies have many implications for human health. For example, one recently discovered target of Smt3-conjugation is the promyelocytic leukemia protein PML. The conjugation of this protein to Smt3 appears to control its localization to discrete nuclear foci termed PML bodies. Recent experiments suggest that Drosophila nuclei may contain similar Smt3-dependent foci.